DNA polymerases with increased 3′-mismatch discrimination

Abstract

Disclosed are mutant DNA polymerases having increased 3′-mismatch discrimination relative to a corresponding, unmodified polymerase. The mutant polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the mutant DNA polymerases.

What is claimed is: 
     
       1. A recombinant nucleic acid encoding a DNA polymerase, wherein the DNA polymerase comprises a motif in the polymerase domain comprising A-G-H-P-F-N-L-N-X 6 -R-D-Q-L-X 10 -R-V-L-X 13 -D-E-L (SEQ ID NO:8), wherein:
 X 6  is S, G, A, D, F, K, C, T, or Y; 
 X 10  is A, G, K, or R; and 
 X 13  is F, A, G, S, T, Y, D, or K, 
 wherein the DNA polymerase has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, or 7. 
 
     
     
       2. An expression vector comprising the recombinant nucleic acid of  claim 1 . 
     
     
       3. A host cell comprising the expression vector of  claim 2 . 
     
     
       4. A method for producing a DNA polymerase, comprising culturing the host cell of  claim 3  under conditions suitable for expression of the recombinant nucleic acid. 
     
     
       5. The recombinant nucleic acid of  claim 1 , wherein X 10  is K. 
     
     
       6. The recombinant nucleic acid of  claim 1 , wherein the DNA polymerase comprises a motif in the polymerase domain comprising A-G-H-P-F-N-L-N-S-R-D-Q-L-X 10 -R-V-L-F-D-E-L (SEQ ID NO:9), wherein:
 X 10  is A, G, K, or R. 
 
     
     
       7. The recombinant nucleic acid of  claim 1 , wherein the DNA polymerase further comprises a motif comprising
 T-G-R-L-S-S-X 7 -X 8 -P-N-L-Q-N; wherein 
 X 7  is Ser (S) or Thr (T); and 
 X 8  is any amino acid other than D or E (SEQ ID NO:27). 
 
     
     
       8. The recombinant nucleic acid of  claim 7 , wherein the amino acid corresponding to position X 8  is selected from the group consisting of L, G, T, Q, A, S, N, R, and K. 
     
     
       9. The recombinant nucleic acid of  claim 8 , wherein the amino acid corresponding to position X 8  is G. 
     
     
       10. The recombinant nucleic acid of  claim 1 , wherein the DNA polymerase has increased 3′-mismatch discrimination activity compared with a control DNA polymerase, wherein the control DNA polymerase has the same amino acid sequence as the DNA polymerase except that the amino acid of the control DNA polymerase corresponding to position 493 of SEQ ID NO:1 is E. 
     
     
       11. A recombinant nucleic acid encoding a thermostable, family A-type DNA polymerase having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, or 7, wherein the amino acid corresponding to position 493 of SEQ ID NO:1 is selected from A, G, K, or R. 
     
     
       12. The recombinant nucleic acid of  claim 11 , wherein the amino acid corresponding to position 580 of SEQ ID NO:1 is G.